This is a proposal to investigate the development of histospecific enzymes in cleavage-arrested embryos (Ciona intestinalis), as a means of observing the segregation of egg cytoplasmic information related to enzyme differentiations (acetylcholinesterase, tyrosinase, and alkaline phosphatase), and as a way of learning something about the possible nature of the information and its interaction with the genome. In addition to further histochemical studies of enzyme distribution, the research will involve a rigorous qualitative and quantitative investigation of the enzymes formed in the cleavage-arrested embryos. Enzyme specificity will be investigated by electrophoretic and immunological techniques. Enzyme quantities will be examined to see how enzyme synthesis is regulated in relation to cell size. Microsurgical techniques will be used to study the possible relationships of inductive tissue interaction in the enzyme expression. The results of actinomycin D inhibition studies have suggested that the egg cytoplasmic information for acetylcholinesterase and tyrosinase differentiations is not mRNA for the enzymes, whereas that for alkaline phosphatase possibly is a preformed maternal mRNA. It is proposed to show by mRNA isolation techniques that actinomycin D prevents mRNA synthesis at all stages of larval development. Isolated mRNA (poly-adenylated RNA) will be used in the in vitro wheat germ derived translation system to determine the time in development when genes for acetylcholinesterase are transcribed, and to show a maternal mRNA for alkaline phosphatase. BIBLIOGRAPHIC REFERENCE: Whittaker, J. R. 1975. Differentiation of two histospecific enzymes in the same cleavage-arrested ascidian egg. Biological Bulletin 149: 451.